Process for producing crystalline nucleus and method of screening crystallization conditions

ABSTRACT

The present invention relates to a process for producing high-quality crystals of protein or organic substances easily and efficiently. A solution of protein or an organic substance is prepared and then is cooled slowly to be supersaturated to a low degree. This supersaturated solution is irradiated with a femtosecond laser  10 . A local explosion phenomenon occurs at the focal point of the laser and thereby a crystalline nucleus is generated. A high-quality crystal is obtained when a crystal is grown on the crystalline nucleus over a long period of time. The femtosecond laser to be used herein can be a titanium:sapphire laser having a wavelength of 800 nm, a duration of 120 fs, a frequency of 1 kHz, and an output of 400 mW.

TECHNICAL FIELD

The present invention relates to a process for producing a crystallinenucleus and a method of screening crystallization conditions.

BACKGROUND ART

With the progress of post-genome studies, it has become necessary toanalyze the protein structure urgently. In order to do that, however, itis necessary to crystallize protein. Furthermore, organic crystals areconsidered as promising next-generation device materials. Accordingly,there are great needs for the technique of producing high-qualityorganic crystals. Generally, in order to deposit crystals from asolution, it is necessary to increase the degree of supersaturationthrough solvent evaporation, a temperature change, etc. However, asubstance with a high molecular weight such as an organic substance orprotein is not crystallized unless the supersaturation degree isextremely high. Once crystallization takes place in such a solutionwhose supersaturation degree is extremely high, the crystal growsrapidly. Hence, there is a possibility that the crystal to be obtainedthereby may have a problem in its quality. Furthermore, such ahigh-molecular-weight substance generally is difficult to crystallize,which results in low productivity. Usually, crystallization conditionsare determined as a result of trial and error through actual attempts ofcrystallization. This, however, is too complicated to be practical inthe case of substances such as protein, an organic substance, etc. thatmust be crystallized. There is an attempt to produce an organic crystalusing a nanosecond Nd:YAG laser (Physical Review Letters 77 (1996)p3475; JP2002-068899). In this method, however, crystallization is notachieved satisfactorily and it is particularly difficult to crystallizeprotein.

DISCLOSURE OF THE INVENTION

The present invention was made in consideration of such situations. Afirst object of the present invention is to provide a technique thatallows high-quality crystals to be produced easily and efficiently. Asecond object of the present invention is to provide a technique thatallows crystallization conditions to be determined easily.

In order to achieve the first object, a process for producing acrystalline nucleus of the present invention is a process for generatinga crystalline nucleus by irradiating a solution in which a solute to becrystallized has dissolved, with at least one pulsed laser selected froma picosecond pulsed laser and a femtosecond pulsed laser.

In this manner, when the solution is irradiated with the pulsed laser, acrystalline nucleus is generated even in a solution that has beensupersaturated to a low degree. Accordingly, a crystal can be grownslowly on the crystalline nucleus. As a result, a high-quality crystalcan be produced easily and efficiently. The process of the presentinvention is most suitable for the crystallization of protein andorganic substances but also can be used for crystallization of othersubstances.

In order to achieve the second object, the method of screeningcrystallization conditions of the present invention is a methodincluding: irradiating a solution in which a solute to be crystallizedhas dissolved, with at least one pulsed laser selected from a picosecondpulsed laser and a femtosecond pulsed laser; and at least one of judgingwhether a crystalline nucleus has been generated by the laserirradiation and judging whether the solute has been altered by the laserirradiation.

In this manner, an observation is made about whether a crystallinenucleus has been generated after the pulsed laser irradiation, andthereby the conditions of the solution, etc. can be judged to besuitable for crystallization if a crystalline nucleus has beengenerated. On the other hand, the state of the solute is observed afterthe pulsed laser irradiation, and thereby the conditions of thesolution, etc. can be judged to be suitable for crystallization if thesolute has altered. In the case of protein, the alteration of the soluteis, for instance, an alteration (denaturation) in three dimensionalstructure.

When a supersaturated solution is irradiated with at least one pulsedlaser selected from a picosecond pulsed laser and a femtosecond pulsedlaser, a crystalline nucleus is generated. The mechanism thereof,however, is unknown. With respect to this, the present inventors presumeas follows. That is, since high-density photons may concentrate at thefocal point of the pulsed laser, a phenomenon (multiphoton absorption)in which several photons collide against one solute molecule or solventmolecule and it then absorbs light occurs with a high probability. As aresult, when the pulsed laser is focused, an explosion phenomenon (laserablation) is induced at the focal point due to the abrupt opticalabsorption. Conceivably, this serves as a perturbation to cause thecrystalline nucleus generation. The following three mechanisms can beconsidered as the mechanism subsequent to this.

-   -   (1) The pulsed laser causes the photothermal conversion to        evaporate the solution instantaneously in the vicinity of the        focal point and thereby the solute is concentrated, which        results in crystalline nucleus generation.    -   (2) Impulse waves are generated by ablation induced by the        pulsed laser, and thereby the solution is rocked locally, which        results in crystalline nucleus generation.    -   (3) Stimulated scattering is caused in the solution when the        laser has an increased pulse energy, and thereby a concentration        gradient is produced in the solution, which results in        crystalline nucleus generation.

As described above, with the present invention, a crystalline nucleuscan be generated easily and efficiently even from protein or an organicsubstance that is difficult to crystallize, and a high-quality crystalcan be obtained when the crystalline nucleus is allowed to grow slowly.Furthermore, with the present invention, crystallization conditions canbe screened and thereby can be determined quickly.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing the configuration of a laser irradiationapparatus used in an example of the present invention.

FIG. 2 is a diagram showing the configuration of an example of theapparatus for measuring the magnitude of impulse waves that aregenerated by laser irradiation.

FIG. 3 is a schematic view showing an example of the relationshipbetween impulse waves that are generated by a pulsed laser and a motionof a particle.

FIG. 4 is a graph showing the relationship between a laser focal pointand a distance for which a particle is moved by the impulse wavesmeasured with the above-mentioned apparatus.

FIG. 5 is a graph showing the relationship between laser pulse energyand the strength of the impulse waves measured with the above-mentionedapparatus.

FIG. 6 is a diagram showing the configuration of a laser irradiationapparatus used in another example of the present invention.

FIG. 7 is a diagram showing the configuration of a laser irradiationapparatus used in still another example of the present invention.

FIG. 8 shows micrographs of protein crystals produced in an example ofthe present invention.

FIG. 9 shows micrographs of protein crystals produced in another exampleof the present invention.

FIG. 10 shows micrographs of protein crystals produced in still anotherexample of the present invention.

FIG. 11 shows micrographs of protein crystals produced in yet anotherexample of the present invention.

FIG. 12 shows micrographs of protein crystals produced in a furtherexample of the present invention.

FIG. 13 shows micrographs of protein crystals produced in still anotherexample of the present invention.

FIG. 14 is a schematic view showing a stirring apparatus used inReference Example 2.

FIGS. 15A and 15B show micrographs of crystals obtained by aconventional method while FIGS. 15C and 15D show micrographs of crystalsobtained in Reference Example 2 described above.

FIG. 16 is a cross sectional view showing an example of the container ofthe present invention.

FIG. 17 is a perspective view showing an example of the plate of thepresent invention.

FIGS. 18A and 18B are a plan view and a cross sectional view showinganother example of the container according to the present invention,respectively.

FIGS. 19A and 19B are a plan view and a cross sectional view showingstill another example of the container according to the presentinvention, respectively.

FIG. 20 is a cross sectional view showing yet another example of thecontainer according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, the present invention is described further in detail.

As described above, in the present invention, it is preferable that atleast one pulsed laser selected from a picosecond pulsed laser and afemtosecond pulsed laser be focused in the solution with a lens to causea single or multiple local explosion phenomena at the focal point andthereby generate a crystalline nucleus. It also is preferable that thesolute be altered by the explosion phenomena.

In the present invention, the pulse peak power (photon flux) of thepulsed laser is, for instance, at least 5×10⁵ (watt), preferably atleast 2×10⁹ (watt). The upper limit of the pulse peak power of thepulsed laser is not particularly limited but is, for instance, nothigher than 10¹⁸ (watt), preferably not higher than 10¹⁵ (watt), andmore preferably not higher than 10¹² (watt).

As described later, since the product of the laser pulse energy (W) andthe duration (Δt) is the pulse peak power (I), the laser conditions canbe set depending on the duration as follows, for example. In this case,a picosecond pulsed laser and a femtosecond pulsed laser can be used asthe pulsed laser, but among them, the femtosecond pulsed laser isparticularly preferable. Picosecond Pulsed Laser Laser Pulse Laser PulsePulse Duration Energy A Energy B (second) (J/pulse) (J/pulse) Common 10⁻⁹-10⁻¹² 0.5 × 10⁻³-0.5 × 10⁻⁶ 2-2 × 10⁻³ Range Preferred 10⁻¹¹-10⁻¹²0.5 × 10⁻⁵-0.5 × 10⁻⁶ 2 × 10⁻²-2 × 10⁻³ RangeLaser Pulse Energy A: Pulse Peak Power (I) = 5 × 10⁵ (watt) or higherLaser Pulse Energy B: Pulse Peak Power (I) = 2 × 10⁹ (watt) or higher

Femtosecond Pulsed Laser Laser Pulse Laser Pulse Pulse Duration Energy AEnergy B (second) (J/pulse) (J/pulse) Common 10⁻¹²-10⁻¹⁵ 0.5 × 10⁻⁶-0.5× 10⁻⁹ 2 × 10⁻³-2 × 10⁻⁶ Range Preferred 10⁻¹³-10⁻¹⁵ 0.5 × 10⁻⁷-0.5 ×10⁻⁹ 2 × 10⁻⁴-2 × 10⁻⁶ RangeLaser Pulse Energy A: Pulse Peak Power (I) = 5 × 10⁵ (watt) or higherLaser Pulse Energy B: Pulse Peak Power (I) = 2 × 10⁹ (watt) or higher

The pulsed laser irradiation may be carried out by a single shot ormultiple repetitive shots. The number of times of the pulsed laserirradiation is not particularly limited but is, for instance, in therange of 1 shot (single shot) to 10 million shots. When the irradiationis carried out by multiple repetitive shots, the laser repetitionfrequency is, for instance, in the range of 1/10000000 Hz to 1 kHz. Theirradiation time also is not particularly limited but is, for instance,in the range of 1 second to 1 hour.

Specific examples of the pulsed laser include a femtosecondtitanium:sapphire laser, a femtosecond fiber laser, ananosecond/picosecond Nd³⁺:YAG laser, a nanosecond/picosecond Nd³⁺:VYO₄laser, an excimer laser, and an alexandrite laser.

In the present invention, the solution in which a solute to becrystallized has dissolved is preferably a supersaturated solution, morepreferably a solution saturated to a low degree. The supersaturation ofa protein solution is, for instance, 200% to 500%, preferably 100% to300%, and more preferably 50% to 200%. The supersaturation of an organicsubstance solution is, for instance, 20% to 50%, preferably 10% to 30%,and more preferably 5% to 20%. Examples of the protein to which thepresent invention can be applied include lysozyme, glucose isomerase,xynalase, myoglobin, catalase, trypsin, human lysozyme, photoactiveyellow protein, phosphoenolpyruvate carboxylase, ribonuclease, aprostaglandin F2 alpha synthetic enzyme, adenosine deaminase, and amultidrug efflux transporter. Examples of the organic substance include4-dimethylamino-N-methyl-4′-N-stilbazolium tosylate (DAST),methylnitroaniline (MNA), and L-arginine phosphate (LAP). As indicatedin examples to be described later, the process of the present inventionmakes it possible to crystallize membrane protein that used to bedifficult to crystallize.

FIG. 1 shows an example of the apparatus used for carrying out theprocess of the present invention. As shown in FIG. 1, this apparatusincludes a femtosecond laser irradiation means 1, a mechanical shutter2, a half-wave plate 3, a polarizer 4, a condenser lens 5, and aconstant-temperature water bath 8. The constant-temperature water bath 8contains water 9, and a sample container 6 is placed therein. The samplecontainer 6 contains a solution 7 in which a solute to be crystallizedhas dissolved. In the constant-temperature water bath 8, the temperatureis lowered gradually to decrease the solubility in the solution 7 andthereby the solution 7 is brought into a supersaturation state. In thisstage, when the solution is supersaturated excessively, crystals growquickly. The solution therefore is supersaturated to a low degree.Subsequently, when emitted from the laser irradiation means 1, a laserbeam 10 passes through the mechanical shutter 2, the half-wave plate 3,the polarizer 4, and the condenser lens 5 to be focused inside thesolution 7. Then an explosion phenomenon is caused at the focal pointdue to abrupt optical absorption to induce crystalline nucleusgeneration. In the case where crystals are to be grown, when they areallowed to grow slowly on crystalline nuclei over a long period of time,high-quality single crystals can be obtained. Furthermore, in the caseof screening crystallization conditions, a plurality of solutions areprepared that are different slightly from each other in concentrationthereof, composition ratio of the solute, or temperature condition.Subsequently, they are irradiated with a laser beam, which then areobserved. When the generation of crystalline nuclei or the alteration ofthe solute is observed, the solution is judged to meet thecrystallization conditions. Otherwise, the solution is judged not tomeet the crystallization conditions. Preferably, the screening method ofthe present invention is used for primary screening for determiningcrystallization conditions.

In the present invention, it is preferable that a container includingthe solution in which a solute to be crystallized has dissolved beallowed to move to stir the solution and thereby a crystal generated andthen grown.

In this manner, when the solution itself is not stirred directly but thecontainer including the solution is allowed to make a movement such as,for instance, rotation, vibration, or rocking to stir the solutionindirectly, gentle stirring can be carried out easily. In addition,since the convection of the solution can be controlled easily, astirring manner that is suitable for the crystal generation can beselected. As a result, it is possible to crystallize high molecules suchas protein that used to be difficult to crystallize.

The above-mentioned movement is not particularly limited. Examplesthereof include rotation, vibration, and rocking, and the movement maybe one in which two or more of them are combined together. The degree ofthe movement also is not particularly limited but is determined suitablyaccording to, for instance, the type of the polymer solution. In thecase of a circular motion, the speed is, for instance, 10 to 1000 rpm,preferably 30 to 200 rpm, and more preferably 50 to 100 rpm.

The container is not particularly limited but can be, for instance, abeaker, a laboratory dish, and a well plate including a plurality ofwells.

Crystals can be generated and grown by, for instance, bringing thesolution into the supersaturation state. The supersaturation state canbe obtained by, for instance, evaporating the solvent contained in thesolution. The evaporation is not particularly limited but can be, forexample, natural evaporation, evaporation by heating, evaporation bydrying under reduced pressure, as well as freezing by lyophilization. Inaddition, another container is prepared that contains a reservoirsolution in which components other than the solute have dissolved athigher concentrations than in the solution. When this container and thecontainer including the solution for crystallization are brought into astate where water vapor can move therebetween, evaporation of thesolution for crystallization can be promoted under gentle conditions.This is preferable particularly for biopolymers such as protein thattend to be denatured. Such a method generally is called a vapordiffusion method.

In the present invention, the place where crystals grow is notparticularly limited. Generally, since grown crystals have a higherspecific gravity than that of the solution, they sink to move to thebottom of the container. Such a method generally is called asitting-drop method. However, when growing in the bottom of thecontainer, the crystals stick thereto, which may cause trouble incollecting them. Hence, it is preferable that a liquid whose specificgravity is higher than that of the solution for crystallization be putin the container and crystals be grown at the interface between thesolution and the liquid with a higher specific gravity. Such a methodgenerally is called a floating-drop method.

In the process in which the container is moved to stir the solution, thesubstance to be crystallized is not particularly limited. Examplesthereof include resin, protein, saccharide, lipid, and nucleic acid.Preferably, this process is applied to biopolymers such as protein thatare particularly difficult to crystallize. Examples of the proteininclude chicken egg white lysozyme, human lysozyme, glucose isomerase,xynalase, phosphoenolpyruvate carboxylase, ribonuclease, a prostaglandinF2 alpha synthetic enzyme, adenosine deaminase, and a multidrug effluxtransporter.

The container of the present invention is a container that is used inthe production process or the screening of the present invention. Thecontainer of the present invention is of the following three types.

A first container of the present invention is a container that is usedin the process for producing a crystalline nucleus or a crystal of thepresent invention or a container that is used in the screening ofcrystallization conditions of the present invention. The containerincludes: a first chamber in which a solution of a substance to becrystallized is put; a second chamber in which a reservoir solution isput, in which only components other than the substance to becrystallized of the solution of the substance to be crystallized havedissolved at higher concentrations than in the solution of the substanceto be crystallized; and a passage that communicates with the firstchamber and the second chamber and allows gas to pass therethrough. Inthe container, a part or the whole of the first chamber is transparentor semitransparent so as to allow the solution of the substance to becrystallized to be irradiated with a laser beam.

With this container, evaporation of the solvent of the solution of thesubstance to be crystallized is promoted by the so-called vapordiffusion method and thereby the generation of crystals of the substanceto be crystallized is promoted. Furthermore, with this container, thefirst chamber containing the solution of the substance to becrystallized is irradiated with a laser beam and thereby crystallinenuclei are generated forcibly or crystallization conditions arescreened.

Furthermore, a plurality of first containers may be formed in one plate.

A second container of the present invention is a container that is usedin the process for producing a crystalline nucleus or a crystal of thepresent invention or a container that is used in the screening ofcrystallization conditions of the present invention. The containerincludes: a first chamber in which a solution of a substance to becrystallized is put; a second chamber in which a reservoir solution isput, in which only components other than the substance to becrystallized of the solution of the substance to be crystallized havedissolved at higher concentrations than in the solution of the substanceto be crystallized; and a passage that communicates with the firstchamber and the second chamber and allows gas to pass therethrough. Thecontainer includes a plurality of first chambers that communicate withat least one second chamber through a plurality of passages. Theplurality of passages are different in at least one of diameter andlength from each other. A part or the whole of the first chamber istransparent or semitransparent so as to allow the solution of thesubstance to be crystallized to be irradiated with a laser beam.

With this container, evaporation of the solvent of the solution of thesubstance to be crystallized is promoted by the so-called vapordiffusion method and thereby the generation of crystals of the substanceto be crystallized is promoted. Furthermore, with this container, sincethe plurality of passages are different in diameter or length or in bothof them from each other, a plurality of vapor diffusion conditions canbe set at a time, which allows optimal crystallization conditions to bescreened or allows crystals to be generated under the optimalcrystallization conditions.

A plurality of second containers may be formed in one plate.

A third container of the present invention is a container that is usedin the process for producing a crystalline nucleus or a crystal of thepresent invention or a container that is used in the screening ofcrystallization conditions of the present invention. The containerincludes: a first chamber in which a solution of a substance to becrystallized and an immiscible hyperbaric solution are put, with theimmiscible hyperbaric solution having a higher specific gravity thanthat of the solution of the substance to be crystallized and beingimmiscible with the solution of the substance to be crystallized; and asecond chamber in which a reservoir solution is put, in which onlycomponents other than the substance to be crystallized of the solutionof the substance to be crystallized have dissolved at higherconcentrations than in the polymer solution. The first chamber is formedin the second chamber. The first chamber includes a large volume part inits lower portion and a small volume part in its upper part, with thesmall volume part having a smaller volume than that of the large volumepart. The end of the upper part is open and gas can pass therethrough tomove between the first and second chambers. The solution of thesubstance to be crystallized is retained in at least the upper part ofthe first chamber or the opening part of the end. A part or the whole ofthe container is transparent or semitransparent so as to allow thesolution of the substance to be crystallized to be irradiated with alaser.

With this container, evaporation of the solvent of the solution of thesubstance to be crystallized is promoted by the so-called vapordiffusion method and thereby the generation of crystals of the substanceto be crystallized is promoted at the interface between the solution ofthe substance to be crystallized and the immiscible hyperbaric solution.Since the solution of the substance to be crystallized is retained inthe small volume part or the opening part of the end of the firstchamber, a small amount of the solution of the substance to becrystallized may be used. Furthermore, when the immiscible hyperbaricsolution located in the large volume part of the first chamber isstirred with, for instance, a magnet stirrer, the solution of thesubstance to be crystallized can be stirred indirectly and therebycrystallization further can be promoted.

With respect to the first chamber of the third container, it ispreferable that the large volume part located in the lower part have areverse truncated cone shape or a reverse truncated pyramid shape, thesmall volume part located in the upper part have a cylindrical shape ora rectangular-cylindrical shape, and the large volume part and the smallvolume part be joined to each other. When the first chamber has such ashape, a droplet of the solution of the substance to be crystallized canbe formed on the opening part of the end of the small volume partlocated in the upper part, and in this state, the solvent of thesolution of the substance to be crystallized can be evaporated.Furthermore, a plurality of third containers may be formed in one plate.

In the present invention, the substance to be crystallized is notparticularly limited. Examples thereof include resin, protein,saccharide, lipid, and nucleic acid. It is preferable that the containerof the present invention be used for crystallizing protein among others.Examples of the protein include chicken egg white lysozyme, humanlysozyme, glucose isomerase, xynalase, phosphoenolpyruvate carboxylase,ribonuclease, a prostaglandin F2 alpha synthetic enzyme, adenosinedeaminase, and a multidrug efflux transporter.

EXAMPLES

Examples of the present invention are described below together withcomparative examples.

Apparatus

The apparatus shown in FIG. 1 was used in Examples 1 and 2, ComparativeExample 1, and reference examples. This apparatus allows a samplesolution 7 contained in a constant-temperature water bath 8 to beirradiated with an intense femtosecond titanium:sapphire laser 10focused by a lens 5 with a focal length of 170 mm. The temperature ofthe constant-temperature water bath 8 can be controlled at an accuracyof ±0.05° C. The laser 10 has a wavelength of 800 nm and a duration of120 fs. The repetition frequency of laser oscillation can be adjustedfrom 1 kHz to 1 Hz. When the sample solution 7 is to be irradiated witha single-shot laser pulse, the repetition frequency is adjusted to 20 Hzand the single-shot pulse is taken out of the pulse train by opening amechanical shutter 2 for only 50 ms. The laser pulse energy can beadjusted with a half-wave plate 3 and a polarizer 4. This apparatusallows the sample solution 7 to be irradiated with a laser pulse whosepulse energy is 250 μJ/pulse (2×10⁹ watt).

Example 1

After 3.5 g of DAST (4-dimetylamino-N-metyl-4′-N-stilbazolium tosylate)were put into a Teflon® container 6 with a volume of 200 ml togetherwith 200 ml of methanol and a rotor, this was placed in aconstant-temperature water bath 8 whose initial temperature was 27.0° C.Thereafter, the temperature was increased to 55.0° C. over two hours andthereby the DAST was dissolved while the solution was stirred with astirrer. After it was ensured that the DAST had dissolved about 5 hourslater, the solution 7 was divided into three, which were used as growthsolutions. In this stage, the rotor was removed. When 16 hours passedafter the preparation of the solutions, the solutions were heated at 55°C. for ten hours and then the temperature was lowered to 23° C. at arate of 3° C./hour. Further, the temperature was lowered to 21.4° C. ata rate of 0.1° C./hour. In this state, the solutions were irradiatedwith a femtosecond laser whose pulse energy was 250 μJ/pulse (2×10⁹watt) at a repetition frequency of 1 kHz for two minutes. Thereafter, nocrystal deposition was observed for one hour. Then crystal depositionwas observed visually ten hours later. The observation was continued forabout six days, with the above-mentioned temperature being maintained.As a result, the crystal that was found first had grown and additionallya few crystals were observed. Then laser irradiation was carried outagain at a pulse energy of 250 μJ/pulse (2×10⁹ watt) and a repetitionfrequency of 1 kHz for one minute. No crystal deposition was observedfor three days after the irradiation.

Example 2

Chicken egg white lysozyme was used as an object from which acrystalline nucleus is to be generated. A solution thereof was preparedas follows. That is, 0.467 g of sodium acetate trihydrate was added to50 ml of distilled water, acetic acid was added thereto so as to adjustit to pH4.5, and then 1.25 g of sodium chloride and 1.25 g of chickenegg white lysozyme were added thereto. The sample solution 7 adjusted toroom temperature was put into a 100-ml Teflon container 6. This was keptin a constant-temperature water bath 8 at 40° C. for 24 hours andthereby complete dissolution was achieved. Thereafter, it was cooled to25° C. over five hours and then impurities were removed therefrom with amembrane filter. Then 2 ml of the solution thus obtained and 3 ml offlorinate were put into each of ten glass bottles 6 with a diameter of18 mm that each were provided with a screw cap. These glass bottles 6were allowed to stand still in the constant-temperature water bath 8whose temperature was kept at 25° C. The temperature of the solution waslowered to 15° C. over 20 hours. The saturation point of the samplesolution (the lysozyme solution) 7 is 23.8° C. Then the solution waschecked and no crystal deposition was observed. Thereafter, thetemperature of the solution was lowered to 14° C. over 24 hours. Thesolution was maintained at 14° C. for 24 hours. It then was checkedagain and thereby no crystal deposition was observed. Thereafter, thesolutions 7 contained in the ten glass bottles were irradiated one byone with a femtosecond pulsed laser whose pulse energy was 250 ΔJ/pulse(2×10⁹ watt) for one minute and thereby changes caused therein wereobserved. With respect to the irradiation conditions, the repetitionfrequency of the laser was varied to 50 Hz and 100 Hz, at each of whichtwo each out of the ten glass bottles were irradiated. Four of the tenglass bottles were not irradiated with the laser to be used as controls.It was observed visually one day after the laser irradiation thatcrystals had deposited in the solutions irradiated at 50 Hz and 100 Hz.No crystal deposition was found in the solutions that had not beenirradiated with the laser. Furthermore, the solutions in which nocrystal deposition had been found were irradiated with a laser at 500 Hzand 1000 Hz. As a result, it was observed that lysozyme had aggregated(denatured) in the solutions. Accordingly, these solutions were judgedto have the possibility of crystallization.

Comparative Example 1

The generation of crystalline nuclei was attempted using a proteinsolution whose conditions were the same as those employed in Example 2,by irradiating the protein solution with a YAG laser (with a wavelengthof 1064 nm, 5 ns). The protein solution was prepared as follows. Thatis, 0.467 g of sodium acetate trihydrate was added to 50 ml of distilledwater, acetic acid was added thereto so as to adjust it to pH4.5, andthen 1.25 g of sodium chloride and 1.25 g of chicken egg white lysozymewere added thereto. The sample solution 7 adjusted to room temperaturewas put into a 100-ml Teflon container. This was kept in aconstant-temperature water bath at 40° C. for 24 hours and therebycomplete dissolution was achieved. Thereafter, it was cooled to 25° C.over five hours and then impurities were removed therefrom with amembrane filter. Then 2 ml of the solution thus obtained and 3 ml offluorinate were put into each of ten glass bottles with a diameter of 18mm that each was provided with a screw cap. These glass bottles wereallowed to stand still in a constant-temperature water bath whosetemperature was kept at 25° C. The temperature of the solution waslowered to 15° C. over 20 hours. The saturation point of the samplesolution (the lysozyme solution) is 23.8° C. Then the solution waschecked and thereby no crystal deposition was observed. Thereafter, thetemperature of the solution was lowered to 14° C. over 24 hours. Thesolution was maintained at 14° C. for 24 hours. It then was checkedagain and thereby no crystal deposition was observed. Thereafter, thesolutions contained in the ten glass bottles were irradiated one by onewith the YAG laser for one minute and thereby changes caused thereinwere observed. With respect to the irradiation conditions, therepetition frequency of the laser was varied to 10 Hz, 20 Hz, 50 Hz, 100Hz, 500 Hz, and 1000 Hz at which six out of the ten glass bottles wereirradiated, respectively. Four of the ten glass bottles were notirradiated with the laser to be used as controls. The laser pulse energywas 1.2 mJ/pulse. No crystal deposition was observed one day after thelaser irradiation. In order to increase the supersaturation degree, thetemperature of the solutions was lowered to 16° C. over 10 hours andthen the solutions were maintained for 12 hours. After they were checkedand thereby no crystal deposition was observed, laser irradiation wascarried out again. The irradiation conditions were the same as thosedescribed above. No crystal deposition was observed again this time.Similarly, in the states where the temperature was adjusted to 15° C.and 14° C., laser irradiation was carried out. However, neither crystaldeposition nor denaturation was observed after all. The laser averagepower employed in this example was ten times higher than that of thefemtosecond pulsed laser used in the examples but the pulse peak power(photon flux) was 2.5×10⁵ (watt), which was 1/10000 of that employed inthe examples. Presumably, this resulted in no generation of crystallinenuclei.

Reference Example 1

Impulse waves that would be caused by the explosion phenomenon producedby a pulsed laser were examined using the apparatus shown in FIG. 2.This apparatus includes an erect microscope and a pulsed laserirradiation apparatus added thereto. As shown in FIG. 2, the erectmicroscope 11 includes a stage 28 on which an object to be observed isplaced, a condenser lens 29, and an objective lens (with a magnificationof 100 times and a numerical aperture of 1.25) 26. A microchip 27 isplaced on the stage 28. A light source lamp 13 is disposed under thecondenser lens 29 located in the lower part of the erect microscope 11.A CCD camera 12 that detects the light of the light source lamp 13 isdisposed in the upper portion of the microscope 11. Furthermore, thepulsed laser irradiation apparatus 21 is disposed outside the erectmicroscope 11. A laser 22 passes through a half-wave plate 23 and apolarizer 24 to reach the inside of the erect microscope 11 that isirradiated therewith. The path of the laser is bent at a right angle bya dichroic mirror 25 and then the inside of the microchip 7 placed onthe stage 28 is irradiated with the laser. The pulsed laser used hereinis an intense femtosecond titanium:sapphire laser (800 nm, 120 fs)employing chirp amplification. In addition, a dispersion liquid ofpolystyrene minute particles (with a diameter of 1 Δm) is contained inthe microchip 27. In this apparatus, when the laser 22 is emitted, it isfocused on the dispersion liquid of polystyrene minute particlescontained in the microchip 27 placed on the stage 28. Thereby the stateof the inside of the microchip 27 is observed with the CCD camera 12.

The effect of impulse waves on the polystyrene minute particles wasexamined using this apparatus. That is, the dispersion liquid ofpolystyrene minute particles was irradiated with a single-shot beam ofthe femtosecond titanium:sapphire laser under the aforementionedconditions. The impulse waves that are presumed to be generated at thefocal point of the laser propagate three-dimensionally. The minuteparticles thereby are pushed away from the focal point of the laser. Themotions of the polystyrene minute particles caused by the impulse waveswere observed with the CCD camera. The distance for which thepolystyrene minute particles moved away from the focal point was checkedand thereby the magnitude of the impulse waves was estimated. FIG. 3 isa schematic view showing the relationship between the force imposed on apolystyrene minute particle and impulse waves generated through pulsedlaser irradiation. In FIG. 3, “f” indicates the force of impulse wavesimposed on the minute particle, “F₀” the force generated by the impulsewaves, “R₀” the initial position of the minute particle, “R” theposition of the minute particle measured from the center of the impulsewaves, “r” the radius of the minute particle, numeral 32 a laser beam,numeral 33 the polystyrene minute particle, numeral 34 the focal pointof the laser, and numeral 37 a dispersion liquid of polystyrene minuteparticles. FIG. 4 is a graph showing the relationship between the pulsedlaser irradiation and the distance for which the minute particle wasmoved by the impulse waves generated by the irradiation. In FIG. 4, thehorizontal axis indicates time, the vertical axis indicates the position(R) of the minute particle measured from the center of the impulsewaves, “R₀” denotes the initial position of the minute particle, “L”denotes the distance for which the minute particle moved, and black dotseach indicate pulsed laser irradiation time. As shown in this graph, itcan be understood that the minute particle instantaneously moves awayfrom the focal point upon the pulsed laser irradiation. When it isassumed that the impulse waves propagate isotropically in threedimensions due to impulse response and the minute particle stops due tothe viscous resistance of water, the following formulae hold.Accordingly, it is possible to estimate the force (F₀) that is generatedby the impulse waves from the relationship between the initial position(R₀) of the minute particle and the distance (L) for which the minuteparticle moved.Force of Impulse Wave imposed on Minute Particle:$f = {F_{0}\frac{\pi\quad r^{2}}{4\pi\quad R_{0}^{2}}{\delta(t)}}$Equation of Motion of Minute Particle:${m\frac{\mathbb{d}^{2}R}{\mathbb{d}t^{2}}} = {{{- 6}\pi\quad\eta\quad r\frac{\mathbb{d}R}{\mathbb{d}t}} + f}$Magnitude of Displacement of Minute Particle:$L = {\left. {{R\left( {t = \infty} \right)} - R_{0}}\Leftrightarrow L \right. = {\frac{F_{0} \cdot r}{24p\quad\eta} \cdot \frac{1}{R_{0}^{2}}}}$

In such a manner, motions of hundreds of minute particles were observedand thereby the relationship between the pulse energy of the femtosecondtitanium:sapphire laser and the force generated by impulse waves wasdetermined. The result is shown in graph in FIG. 5. In this graph, thevertical axis indicates the force (F₀) of impulse waves while thehorizontal axis indicates the laser pulse energy (I_(o)). As shown inFIG. 5, impulse waves were observed at 60 nJ/pulse or higher in thisreference example. The threshold of the explosion phenomenon depends onthe probability of multiphoton absorption. Accordingly, the threshold isdefined by not the laser pulse energy that is defined as the total laserenergy but the pulse peak power (I) of a beam that reachesinstantaneously. In this reference example, since the laser pulse energy(W) of the pulsed laser with a duration (Δt) of 120 fs was 60 nJ/pulse,the pulse peak power (I) of the laser beam is calculated as follows:I=W/Δt=5×10⁵ (J/s·pulse=watt).

Hence, the duration (Δt) is expressed as Δt<W/5×10⁵ in this referenceexample. With consideration given to this formula and the performance ofthe laser irradiation apparatus, the pulsed laser preferably has aduration of nanoseconds or shorter, more preferably picoseconds orshorter, and most suitably femtoseconds or shorter. The presentinvention, however, is not interpreted limitedly by this referenceexample. In this reference example, it is assumed that the explosionphenomenon occurs at the focal point of the laser and thereby impulsewaves are generated. The present invention, however, is not limitedthereto.

In this reference example, polystyrene minute particles were used tovisualize impulse waves. However, the polystyrene minute particles eachhas a weight of 1.1×10⁻¹² g, which is at least 100 million times of theweight of protein. That is, there is a possibility that protein may bechanged in its density even by impulse waves that are far weaker ascompared to the polystyrene minute particles. The laser pulse energy andthe pulse peak power of the pulsed laser defined through thevisualization of the polystyrene minute particles do not limit the lowerlimits thereof in the present invention.

Example 3

This example is an example of crystallization of chicken egg whitelysozyme (14 kDa). A protein solution was prepared at room temperatureunder the following conditions. That is, it included 25 mg/ml oflysozyme, 2.5% of sodium chloride, and 0.1 M of sodium acetate, and ithad a pH value of 4.5. Impurities were removed from this solution usinga membrane filter.

The crystallization was carried out by a batch method using a laserirradiation apparatus and a crystallization plate shown in FIG. 6. Asshown in FIG. 6, in this laser irradiation apparatus, a laser emittedfrom a femtosecond laser irradiation apparatus 101 passes through amirror 102, a wavelength plate 103, polarizers 104, and a shutter 105and then is focused with an objective lens (with a magnification of 10times) 108. A crystal growth vessel part 107 of the crystallizationplate 109 then is irradiated therewith. The crystallization plate 109allows the vapor diffusion method to be utilized and includes crystalgrowth vessel parts 107 in which the protein solution is put and serversolution parts 106 that communicate with the crystal growth vessel parts107, respectively. The server solution is a solution in which the samecomponents as those of the protein solution other than protein havedissolved. This promotes evaporation of the protein solution. In FIG. 6,numeral 110 indicates a sealing tape while numeral 111 denotes atransparent glass. The crystal growth vessel parts 107 can be observedvisually with an ocular lens 112 or can be observed using a CCD 113camera and a monitor 114.

The laser irradiation was carried out in a clean room (with atemperature of 23° C.±2° C. and a humidity of 65%±5%) whose temperatureand humidity were controlled. The wavelength of the laser was 780 nm,the duration thereof was 200 femtoseconds (fs), and the repetitionfrequency of laser oscillation was 1 kHz. The laser pulse energy wasadjusted with the wavelength plate and the polarizers. The number oftimes of the laser beam irradiation was adjusted by changing the periodof time for which the shutter was opened and closed.

After 10 μl of protein solution were put into each crystal growth vesselpart 107 of the crystallization plate 110, the solution was irradiatedwith a focused laser beam at room temperature. The laser pulse energywas 1.95 nJ/pulse. The number of pulses was varied to 8 pulses ( 1/125second), 62 pulses ( 1/16 second), 24000 pulses (24 seconds), and zeropulse, i.e. no irradiation, (zero second) by adjusting the period oftime for which the shutter was opened.

After the laser irradiation, it was allowed to stand still in a constanttemperature bath whose temperature had been set at 18° C. and thecrystal growth thereafter was observed. FIG. 8 shows micrographs takentwo days after the laser irradiation. The number of crystals thatdeposited increased with the increase in the number of times of laserirradiation. Furthermore, in the sample that was not subjected to thelaser irradiation, a large number of small crystals deposited. This isbecause the solution had been highly supersaturated and therebycrystalline nuclei were generated spontaneously all over the solution,which resulted in the generation of a large number of nuclei that wascaused at a time. This often is observed in usual crystal growth and isa barrier to the improvement in quality and the increase in size ofcrystals. On the other hand, in the samples that had been irradiatedwith the laser, crystalline nuclei were generated in a solutionsupersaturated to a low degree that had a room temperature, and thenuclei then served as seed crystals and grew. Hence, the increase in thenumber of pulses resulted in the increase in the number of crystallinenuclei to be generated and in turn, the number of deposited crystalsincreased.

Thus, it is conceivable that the generation of crystalline nuclei is aphenomenon depending on the number of times of laser pulse irradiation.The increase in the number of crystals that is caused by the increase inthe number of times of irradiation means the increase in probability ofcrystal generation that is caused by the increase in the number of timesof irradiation. Furthermore, the phenomenon (an incubation effect)generally has been known that the laser pulse energy to be required forcausing the explosion phenomenon by a laser decreases with the increasein the number of times of irradiation (for instance, S. Preuss et al.(Appl. Phys. Lett. 62(23), 7 Jun. 1993, p 3049-3051)). In other words,it is conceivable that the increase in the number of times ofirradiation results in decreases in laser pulse energy and pulse peakpower of laser pulses to be required for crystal generation. In thisexample, the laser pulse energy provided by multiple shots of laserirradiation is much lower than the lower limit of the laser pulse energydefined by the motion of a polystyrene minute particle. Conceivably,this is because of the size of the protein molecule that is far smallerthan the polystyrene minute particle and the effect of the multipleshots of pulsed laser irradiation. These, however, are mere surmises ofthe present inventors and therefore do not limit the scope of thepresent invention.

Example 4

This example is an example of crystallization of ribonuclease H (17kDa). A protein solution was prepared by dissolving 5 mg/ml ofribonuclease H in 0.05 M of Tris hydrochloric acid buffer solution (pH9.0) at room temperature. Thereafter, impurities were removed therefromwith a membrane filter. In addition, 0.2 M of Tris hydrochloric acidwith a pH value of 9.0 was prepared as a server liquid (an externalliquid). The crystallization to be implemented by laser irradiation wascarried out by the sitting-drop vapor diffusion method using the laserirradiation apparatus and the crystallization plate shown in FIG. 6.

That is, 10 μl of protein solution were put into each crystal growthvessel part 107 while 100 μl of external liquid were poured into eachserver solution part 106. Then the protein solution was irradiated witha focused laser beam at room temperature. The laser irradiation wascarried out in a clean room (with a temperature of 23° C.±2° C. and ahumidity of 65%±5%) whose temperature and humidity were controlled. Thewavelength of the laser was 780 nm, the duration thereof was 200femtoseconds (fs), and the repetition frequency of laser oscillation was1 kHz. The laser pulse energy was adjusted with the wavelength plate andthe polarizers. The number of times of the laser beam irradiation wasadjusted by changing the period of time for which the shutter was openedand closed. The laser pulse energy was 1.95 nJ/pulse. The number ofpulses was varied to 8 pulses ( 1/125 second), 62 pulses ( 1/16 second),4000 pulses (4 seconds), 8000 pulses (8 seconds), and zero pulse, i.e.no irradiation (zero second), by adjusting the period of time for whichthe shutter was opened.

After the laser irradiation, it was allowed to stand still in a constanttemperature bath whose temperature had been set at 14° C. and thecrystal growth thereafter was observed. FIG. 9 shows micrographs takenone day after the laser irradiation. In the sample that was notsubjected to the laser irradiation, crystal deposition was not observedeven after two weeks elapsed. However, the differences in depositionstate were observed that were caused depending on the laser irradiationconditions. No crystalline nuclei were generated in the solution thatwas irradiated at 8 pulses while crystal deposition was observed in thesolutions that were irradiated at 62 pulses and 4000 pulses.Furthermore, denatured ribonuclease H was found in the solution that hadbeen irradiated at 8000 pulses.

Example 5

This example is an example of crystallization of glucose isomerase (173kDa). A protein solution was prepared by dissolving 20 mg/ml of glucoseisomerase in 0.2 M of ammonium sulfate solution (pH 7.0) at roomtemperature. Thereafter, impurities were removed therefrom with amembrane filter. In addition, a solution (pH 7.0) in which 0.2 M ofammonium sulfate and 15% of polyethylene glycol (PEG) 6000 weredissolved was prepared as a server liquid (an external liquid). Thecrystallization to be implemented by laser irradiation was carried outby the sitting-drop vapor diffusion method using the laser irradiationapparatus and the crystallization plate shown in FIG. 6.

That is, 10 μl of protein solution were put into each crystal growthvessel part 107 while 100 μl of external liquid were poured into eachserver solution part 106. Thereafter, 5 μl of external liquid werepipetted from the server solution part 106 and then were poured into thecrystal growth vessel part 107. Then the protein solution and the serversolution were mixed together well, which then was irradiated with afocused laser beam at room temperature. The laser irradiation wascarried out in a clean room (with a temperature of 23° C.±2° C. and ahumidity of 65%±5%) whose temperature and humidity were controlled. Thewavelength of the laser was 780 nm, the duration thereof was 200femtoseconds (fs), and the repetition frequency of laser oscillation was1 kHz. The laser pulse energy was adjusted with the wavelength plate andthe polarizers. The number of times of the laser beam irradiation wasadjusted by changing the period of time for which the shutter was openedand closed. The laser pulse energy was 1.95 nJ/pulse. The number ofpulses was varied to 8 pulses ( 1/125 second), 4000 pulses (4 seconds),and zero pulse, i.e. no irradiation (zero second) by adjusting theperiod of time for which the shutter was opened.

After the laser irradiation, it was allowed to stand still in a constanttemperature bath whose temperature had been set at 18° C. and thecrystal growth thereafter was observed. FIG. 10 shows micrographs takenone day after the laser irradiation. In the solutions that wereirradiated with the laser, crystal deposition was observed. In thesample that was not subjected to the laser irradiation, however, crystaldeposition was not observed even after one month elapsed. In thisexample, glucose isomerase that was a huge protein with a molecularweight of about 200000 was crystallized. Hence, the process of thepresent invention using laser irradiation also is effective forcrystallization of such a huge protein.

Example 6

This example is an example of crystallization of a Trypanosoma-derivedprostaglandin F2 alpha synthetic enzyme (31 kDa). A protein solution wasprepared by dissolving 20 mg/ml of the above-mentioned synthetic enzymeand 0.005 M of nicotinamide adenine dinucleotide phosphate (NADP+) wellin 0.04 M of Tris hydrochloric acid buffer solution (pH 8.0) at roomtemperature. Thereafter, impurities were removed therefrom with amembrane filter. In addition, a solution (pH 7.5) containing 0.01 M ofHepes sodium hydroxide buffer solution (HEPES-NaOH), 2% of polyethyleneglycol (PEG) 400, and 1.2 M of ammonium sulfate was prepared as a serverliquid (an external liquid).

The crystallization to be implemented by laser irradiation was carriedout by the hanging-drop vapor diffusion method using the laserirradiation apparatus and the crystallization container shown in FIG. 7.As shown in FIG. 7, in this laser irradiation apparatus, a laser emittedfrom the femtosecond laser irradiation apparatus 101 passes through amirror 102, a wavelength plate 103, polarizers 104, and a shutter 105and then is focused with an objective lens (with a magnification of 10times) 108. A crystal solution 117 contained in a crystallizationcontainer 118 then is irradiated therewith. The crystal solution 117 canbe observed visually with an ocular lens 112 or can be observed using aCCD 113 camera and a monitor 114. In FIG. 7, numeral 116 denotes anexternal liquid, numeral 115 indicates a glass plate, and numeral 119denotes grease.

As shown in FIG. 7, a droplet of the crystallization solution containing2 μl of protein solution and 2 μl of external liquid that had been mixedtogether was formed on the glass plate 115. Thereafter, 500 μl ofexternal liquid 116 were put into the crystallization container 118 andthe crystallization container 118 was covered with the glass plate, withthe droplet 117 of the crystallization solution hanging from the glass.In this case, the grease 119 was used for sealing the crystallizationcontainer 118. The crystallization container 118 then was turned upsidedown while caution was taken to prevent the external liquid 116 fromdropping. Subsequently, the droplet 117 of the crystallization solutionwas irradiated with a focused laser beam. The laser irradiation wascarried out in a clean room (with a temperature of 23° C.±2° C. and ahumidity of 65%±5%) whose temperature and humidity were controlled. Thewavelength of the laser was 780 nm, the duration thereof was 200femtoseconds (fs), and the repetition frequency of laser oscillation was1 kHz. The laser pulse energy was adjusted with the wavelength plate andthe polarizers. The number of times of the laser beam irradiation wasadjusted by changing the period of time for which the shutter was openedand closed. The laser pulse energy was 1.95 nJ/pulse. The irradiationwas carried out at 62 pulses ( 1/16 second) by adjusting the period oftime for which the shutter was opened. A sample that had not beensubjected to the laser irradiation was grown as a control experiment.

After the laser irradiation, it was allowed to stand still in a constanttemperature bath whose temperature had been set at 20° C. and thecrystal growth thereafter was observed. FIG. 11 shows micrographs takenseven days after the laser irradiation. In the solution that had beenirradiated with the laser, crystal deposition was observed. In thesample that had not been subjected to the laser irradiation, however,crystal deposition was not observed even after three months elapsed. Inthis example, the laser irradiation allowed crystals to be produced in ashort period of time.

Example 7

This example is an example of crystallization of adenosine deaminase(ADA) native. A protein solution was prepared by dissolving 20 mg/ml ofADA native in 0.0025 M of Hepes buffer solution (pH 7.5) at roomtemperature. Thereafter, impurities were removed therefrom with amembrane filter. In addition, the following two solutions were preparedas server solutions (external liquids): (1) a solution (pH 6.5)containing 0.2 M of sodium citrate, 0.1 M of sodium cacodylate, and 30%of isopropanol; and (2) a solution (pH 5.6) containing 0.2 M of ammoniumacetate, 0.1 M of sodium citrate, and 30% of polyethylene glycol (PEG)4000.

The crystallization to be implemented by laser irradiation was carriedout by the sitting-drop vapor diffusion method using the laserirradiation apparatus and the crystallization plate shown in FIG. 6.First, 2 μl of protein solution were put into each crystal growth vesselpart 107 while 100 μl of each of the external liquids (1) and (2) werepoured into each server solution part 106. Thereafter, 2 μl of externalliquids were pipetted from the server solution part 106 and then werepoured into the crystal growth vessel part 107. The protein solution andthe external liquids were mixed together well, which then was irradiatedwith a focused laser beam at room temperature. The laser pulse energywas 1.95 nJ/pulse. The irradiation was carried out at 1000 pulses (1second) by adjusting the period of time for which the shutter wasopened. A sample that had not been subjected to the laser irradiationwas grown as a control experiment.

After the laser irradiation, it was allowed to stand still in a constanttemperature bath whose temperature had been set at 20° C. and thecrystal growth thereafter was observed. FIG. 12 shows micrographs takenseven days after the laser irradiation. In the solution that had beenirradiated with the laser, crystal deposition was observed. In thesample that had not been subjected to the laser irradiation, however,crystal deposition was not observed even after one month elapsed.Crystals of the present protein had never been obtained before, but thepresent example made it possible to crystallize the protein for thefirst time.

Example 8

This example is an example of crystallization of multidrug effluxtransporter (AcrB) contained in Escherichia coli. The AcrB is apolytopic-type membrane protein, more specifically a trimer protein inwhich three monomers composed of 1049 amino acid residues are entangled.

A protein solution was prepared by adding 0.02 M of sodium phosphate (pH6.2), 10% of glycerol, and 0.2% of dodecyl maltoside to 28 mg/ml of AcrBlabeled with histidine. A server liquid (an external liquid) wasprepared by blending 14.1% to 14.6% of polyethylene glycol (PEG) 4000,0.08 M of sodium phosphate (pH 6.2), and 0.02 M of sodiumcitrate-hydrochloric acid (pH 5.6).

The crystallization to be implemented by laser irradiation was carriedout by the sitting-drop vapor diffusion method using the crystallizationcontainer and the apparatus shown in FIG. 6.

That is, 2 μl of protein solution were put into each crystal growthvessel part 107 while 50 μl of external liquid described above werepoured into each server solution part 106. Thereafter, 2 μl of externalliquid were pipetted from the server solution part 106 and then werepoured into the crystal growth vessel part 107. Thus the proteinsolution and the external liquid were mixed together well. In thisexample, six growth solutions were prepared that have concentrations ofPEG 4000 varying from 14.1% to 14.6% in increments of 0.1%,respectively. Crystallization was carried out using the respectivegrowth solutions. Thereafter, each protein solution was irradiated withan intense femtosecond titanium:sapphire laser that was focused with anobjective lens (with a magnification of 10 times). The irradiation wascarried out in a clean room (with a temperature of 23° C.±2° C. and ahumidity of 65%±5%) whose temperature and humidity were controlled. Thewavelength of the laser was 780 nm, the duration thereof was 200femtoseconds (fs), and the repetition frequency of laser oscillation was1 kHz. The laser pulse energy was 800 nJ/pulse. The irradiation wascarried out at 250 pulses (¼ second) by adjusting the period of time forwhich the shutter was opened. A sample that had not been subjected tothe laser irradiation was grown as a control experiment.

After the laser irradiation, they were allowed to stand still in aconstant temperature bath whose temperature had been set at 25° C. andthe crystal growth thereafter was observed. FIG. 13 shows micrographstaken two days after the laser irradiation. As shown in FIG. 13, in allthe growth solutions that had been irradiated with the laser, crystaldeposition was observed (indicated with arrows in FIG. 13) although thecrystal growth solutions were prepared with concentrations ofpolyethylene glycol (PEG) 4000 varying from each other. On the otherhand, in the sample that had not been subjected to the laserirradiation, crystal deposition was not observed even when one weekelapsed (not shown in FIG. 13).

Membrane protein is difficult to crystallize, but crystallization ofmembrane protein was achieved in this example.

Reference Example 2

This reference example is an example of crystallization of protein thatwas carried out by moving a container.

A solution was stirred in growing crystals of hen egg white lysozyme. Aprotein solution used herein was prepared as follows. That is, aceticacid was added to 50 ml of distilled water and 0.467 g of sodium acetatetrihydrate so that the solution was adjusted to pH 4.5, and then 1.25 gof sodium chloride and 1.25 g of hen egg white lysozyme were addedthereto. The solution thus prepared at room temperature was put into a100-ml Teflon container and then was maintained in aconstant-temperature water bath at 40° C. for 24 hours. Thereby completedissolution was achieved. Thereafter, it was cooled to 25° C. over fivehours and then impurities were removed therefrom with a membrane filterhaving holes with a diameter of 0.2 μm. On the other hand, a reservoirsolution used herein was prepared as follows. That is, acetic acid wasadded to 50 ml of distilled water and 0.467 g of sodium acetatetrihydrate so that the solution was adjusted to pH 4.5, and then 3 g ofsodium chloride were added thereto.

Using 300 μl of reservoir solution with respect to 3 μl and 10 μl ofprotein solutions, the difference caused depending on whether thesolution was stirred was examined. The crystallization method usedherein was the sitting-drop vapor diffusion method. In addition, theexperiment also was carried out by the floating-drop vapor diffusionmethod in which fluorinate (a solution with a higher specific gravitythan that of the protein solution) was used and crystals were grown atthe interface between the two liquids. The amount of fluorinate solutionwas 10 μl. The crystal growth was carried out at a constant temperature,20° C.

A rotary shaker (BR-15, manufactured by TIETECH CO., LTD) was used as astirring mechanism. The rotational speed was set at 50 rpm at which thesolution was stirred gently. FIG. 14 shows a schematic view of thisstirring apparatus. As shown in FIG. 14, well plates 201 were placed ona shaker 202 and subjected to rotary motion. Thus the solution wasstirred. Each well of the well plates 201 is composed of two small wellsand one large well. The small wells that are subjected to thefloating-drop method contain the protein solutand fluorinate 214 whilethose that are subjected to the sitting-drop method contain the proteinsolution 212. On the other hand, a large well contains a reservoirsolution 213 including components other than lysozyme that havedissolved at high concentrations. This method allows a large amount ofsolution to be stirred easily at a time.

FIG. 15 shows the results of crystal growth. Clear differences in thenumber of deposited crystals and crystal size were caused depending onwhether the solution was stirred. As shown in FIGS. 15A and 15B, in theconventional method in which the solution was not stirred, a number ofvery small crystals (microcrystals) were deposited. On the other hand,as shown in FIGS. 15C and 15D, when the solution was stirred duringcrystal growth, a smaller number of crystals deposited and largercrystals were obtained. Furthermore, as compared to thesolution-stirring sitting-drop method (see FIG. 15C), thesolution-stirring floating-drop method (FIG. 15D) allowed a smallernumber of crystals to deposit and crystals to have larger sizes.

Next, examples of the container according to the present invention aredescribed with reference to the drawings.

Example 9

An example of the first container according to the present invention isshown in the cross sectional view in FIG. 16. As shown in FIG. 16, thiscontainer 301 includes a first chamber 311 and a second chamber 313 thatcommunicate with each other through a passage. The top of this container301 is covered with a lid 316 and thereby the container 301 is in thesealed state. The portion of the bottom part 315 of the container 301that corresponds to the first chamber is transparent or semitransparentso that a laser beam 317 can pass therethrough. The material of theportion of the bottom is not particularly limited as long as ittransmits a laser beam. Examples of the material to be used hereininclude transparent members such as silica glass, glass, transparentresin such as, for example, acrylic resin. The material of the partsother than the portion of the bottom is not particularly limited but,for instance, common resins, glass, etc. can be used. The size of thewhole container is not particularly limited but is, for instance, L 20to 180 mm×W 10 to 120×H 3 to 50 mm, preferably L 40 to 150 mm×W 20 to100×H 5 to 30 mm, and more preferably L 50 to 130 mm×W 30 to 80×H 10 to20 mm. A solution 312 such as, for instance, a protein solution is putin the first chamber 311 while a reservoir solution 314 is put in thesecond chamber 313. Accordingly, it is preferable that the first chamber311 be smaller than the second chamber 313. This container 301 may beused as follows, for example.

First, after the lid 316 is removed, a polymer solution 312 such as, forinstance, a protein solution is put into the first chamber 311 while areservoir solution 314 is put into the second chamber 313. The containerthen is covered with the lid 316 to be sealed. Then, as indicated withan arrow in FIG. 16, water vapor generated from the polymer solution 312moves to the second chamber 313 through the passage. This promotesevaporation of the solvent of the polymer solution 312. When the polymersolution 312 is brought into a supersaturation state, the polymersolution 312 is irradiated with, for instance, a pulsed laser beam 317from the side of the bottom of the container 301 in order to generatecrystalline nuclei forcibly. If crystalline nuclei have been generated,crystals then are allowed to grow thereafter. On the other hand, if nocrystalline nuclei have been generated, the conditions employed therebyare judged to be unsuitable for crystallization and then crystallizationis attempted under other conditions.

As described above, when pulsed laser irradiation is carried out andobservations are made to judge whether crystalline nuclei have beengenerated, if crystalline nuclei have been generated, the conditions ofthe solution, etc. can be judged to be suitable for crystallization.When crystal growth then is carried out thereafter, crystals can beobtained. Furthermore, pulsed laser irradiation is carried out and thestate of the solute then is observed. If the solute has altered, theconditions of the solution, etc. can be judged to be suitable forcrystallization. In the case of protein, the alteration of the solutecan be an alteration (denaturation) in three dimensional structure, forinstance.

With respect to the container of the present invention, the type oflaser, irradiation conditions, etc. are the same as those indicated in,for instance, the description of the production process according to thepresent invention. These laser conditions, etc also are common to othercontainers or plates of the present invention.

Example 10

Next, an example in which a plurality of the above-mentioned firstcontainers are formed in one plate is shown in the perspective view inFIG. 17.

As shown in FIG. 17, this plate 302 includes six first containers 321formed therein. Each of the first containers 321 includes a firstchamber 321 a and a second chamber 321 b that communicate with eachother through a passage. This plate 302 is composed of a plate body 322in which the first containers 321 are formed, a bottom part 323, and alid 324. The bottom part 323 is formed of a transparent orsemitransparent member so as to allow irradiation of a laser beam 325 tobe carried out. The materials, of which the plate 302 is formed, thesize of the containers 321, etc. are the same as in the first containerdescribed above. The size of this plate 302 is not particularly limitedbut is, for instance, L 20 to 180 mm×W 10 to 120×H 3 to 50 mm,preferably L 40 to 150 mm×W 20 to 100×H 5 to 40 mm, and more preferablyL 50 to 130 mm×W 30 to 80×H 10 to 30 mm. The number of containers 321 issix in this plate 302, but the present invention is not limited thereto.It may be 1 to 1536, preferably 2 to 384, and more preferably 4 to 96per plate. This plate 302 is used as follows, for example.

That is, first, a polymer solution and a reservoir solution are put intothe first chamber 321 a and the second chamber 321 b of the container321, respectively, and thereby vapor diffusion occurs to promoteevaporation of the solvent of the polymer solution. After the polymersolution is brought into a supersaturation state, the irradiation of thepulsed laser 325 is carried out as described before and therebycrystalline nuclei are generated forcibly. When crystalline nuclei havebeen generated, they are allowed to grow and thereby intended polymercrystals can be obtained. On the other hand, when no crystalline nucleihave been generated, the crystallization conditions used thereby arejudged to be inappropriate, and crystallization is attempted under thenext conditions. This plate includes a plurality of containers formedtherein. Hence, crystallization conditions that are different from eachother in, for instance, concentration of the polymer solution can be setin the respective containers. Furthermore, the respective containers canbe irradiated with a laser under different conditions.

Example 11

An example of the second container according to the present invention isshown in FIG. 18. In FIG. 18, FIG. 18A is a plan view while FIG. 18B isa cross sectional view.

As shown in FIGS. 18A and 18B, a container 303 has a shape in which acylinder is joined to the bottom of a disk. In the peripheral portion ofthe disk, eight first chambers 331 are formed radially from the circlecenter. The cylinder has one second chamber 332 formed therein. Thefirst chambers 331 communicate with the second chamber 332, withpassages 333 extending from the respective first chambers 331 to thesecond chamber 332. The eight passages 333 are different in diameterfrom each other. The size of the whole container 303 is not particularlylimited. It is determined suitably according to the size and the numberof the first chambers, the size of the second chamber, etc. With respectto the size of the first chambers, each of them has, for instance, aninner diameter of 0.5 to 10 mm and a depth of 1 to 50 mm, preferably aninner diameter of 1 to 5 mm and a depth of 3 to 40 mm, and morepreferably an inner diameter of 1 to 3 mm and a depth of 3 to 30 mm. Thenumber of the first chambers is, for instance, 1 to 1536, preferably 2to 384, and more preferably 4 to 96. With respect to the size of thesecond chamber, it has, for instance, an inner diameter of 1 to 30 mmand a depth of 1 to 50 mm, preferably an inner diameter of 2 to 20 mmand a depth of 2 to 40 mm, and more preferably an inner diameter of 3 to15 mm and a depth of 3 to 30 mm. The length of the passages also is notlimited but is, for instance, 0.5 to 50 mm, preferably 1 to 30 mm, andmore preferably 1 to 20 mm. These passages are different in diameterfrom each other. The diameter is, for instance, 0.3 to 10 mm, preferably0.5 to 5 mm, and more preferably 0.5 to 3 mm. Furthermore, the materialof the container 303 also is not particularly limited but it may beformed of resin or glass, for example. However, when the first chamberis to be irradiated with a laser, it is advantageous that the portionthrough which laser irradiation is to be carried out is formed of thetransparent or semitransparent member described before so as to transmitthe laser. This container 303 is used as follows, for example.

That is, first, a polymer solution 334 and a reservoir solution 335 areput into the plurality of first chambers 331 and the second chamber 332,respectively. As indicated with arrows in FIG. 18B, water vaporgenerated from the polymer solution 334 then passes through the passages333 to move to the second chamber 332. This vapor diffusion promotesevaporation of the solvent of the polymer solution 334. Since thepassages 333 are different in diameter from each other, the vapordiffusion conditions are different in the respective first chambers 333.Accordingly, when crystalline nuclei have been generated in a pluralityof first chambers, they are allowed to grow thereafter and therebyintended polymer crystals can be obtained. On the other hand, withrespect to first chambers in which no crystalline nuclei have beengenerated, the crystallization conditions employed therein can be judgedto be unsuitable. Furthermore, after the polymer solution 334 is broughtinto a supersaturation state, crystalline nuclei may be generatedforcibly by pulsed laser irradiation as described before. Whencrystalline nuclei have been generated, they are allowed to grow andthereby intended polymer crystals can be obtained. On the other hand,when no crystalline nuclei have been generated, the crystallizationconditions used thereby are judged to be inappropriate, andcrystallization is attempted under the next conditions.

Example 12

Next, another example of the second container according to the presentinvention is shown in FIG. 19. In FIG. 19, FIG. 19A is a plan view whileFIG. 19B is a cross sectional view.

As shown in FIGS. 19A and 19B, the container 304 has a shape in which acylinder is joined to the bottom of a disk. In the peripheral portion ofthe disk, eight first chambers 341 are formed radially from the circlecenter. The cylinder has one second chamber 342 formed therein. Thefirst chambers 341 communicate with the second chamber 342, withpassages 343 extending from the respective first chambers 341 to thesecond chamber 342. The eight passages 343 are different in length fromeach other. The size of the whole container 304 is not particularlylimited. It is determined suitably according to the size and the numberof the first chambers, the size of the second chamber, etc. With respectto the size of the first chambers, each of them has, for instance, aninner diameter of 0.5 to 10 mm and a depth of 1 to 50 mm, preferably aninner diameter of 1 to 5 mm and a depth of 3 to 40 mm, and morepreferably an inner diameter of 1 to 3 mm and a depth of 3 to 30 mm. Thenumber of the first chambers is, for instance, 1 to 1536, preferably 2to 384, and more preferably 4 to 96. With respect to the size of thesecond chamber, it has, for instance, an inner diameter of 1 to 30 mmand a depth of 1 to 50 mm, preferably an inner diameter of 2 to 20 mmand a depth of 2 to 40 mm, and more preferably an inner diameter of 3 to15 mm and a depth of 3 to 30 mm. The length of the passages also is notlimited but is, for instance, 0.5 to 50 mm, preferably 1 to 30 mm, andmore preferably 1 to 20 mm. These passages are different in diameterfrom each other. The diameter is, for instance, 0.3 to 10 mm, preferably0.5 to 5 mm, and more preferably 0.5 to 3 mm. Furthermore, the materialof the container 4 also is not particularly limited but it may be formedof resin or glass, for example. However, when the first chamber is to beirradiated with a laser, it is advantageous that the portion throughwhich laser irradiation is to be carried out is formed of thetransparent or semitransparent member described before so as to transmitthe laser. This container 304 is used as follows, for example.

That is, first, a polymer solution 344 and a reservoir solution 345 areput into the plurality of first chambers 341 and the second chamber 342,respectively. As indicated with arrows in FIG. 19B, water vaporgenerated from the polymer solution 344 then passes through the passages343 to move to the second chamber 342. This vapor diffusion promotesevaporation of the solvent of the polymer solution 344. Since thepassages 343 are different in length from each other, the vapordiffusion conditions are different in the respective first chambers 343.Accordingly, when crystalline nuclei have been generated in a pluralityof first chambers, they are allowed to grow thereafter and therebyintended polymer crystals can be obtained. On the other hand, withrespect to first chambers in which no crystalline nuclei have beengenerated, the crystallization conditions employed therein can be judgedto be unsuitable. Furthermore, after the polymer solution 344 is broughtinto a supersaturation state, crystalline nuclei may be generatedforcibly by pulsed laser irradiation as described before. Whencrystalline nuclei have been generated, they are allowed to grow andthereby intended polymer crystals can be obtained. On the other hand,when no crystalline nuclei have been generated, the crystallizationconditions used thereby are judged to be inappropriate, andcrystallization is attempted under the next conditions.

The diameters of the passages were varied in Example 11 while thelengths of the passages were varied in Example 12. However, acombination thereof may be employed. Furthermore, a plurality of atleast one of the container according to Example 11, the containeraccording to Example 12, and a container that is a combination thereofmay be formed in one plate. The conditions such as the size of thisplate, etc. are the same as in, for instance, the plate of Example 10.

Example 13

An example of the third container according to the present invention isshown in FIG. 20. As shown in FIG. 20, this container 305 has aconfiguration in which a small container 352 is disposed in a largecontainer 351. The large container 351 has a cylindrical shape and thetop thereof is covered with a lid. The small container 352 is formed ofa body part of a reverse truncated cone shape (a large volume part) anda cylindrical part (a small volume part) joined to the upper part of thebody part. The end of said cylindrical part is open. The space formedbetween the inner wall of the large container 351 and the outer wall ofthe small container 352 is a second chamber in which a reservoirsolution 354 is put. The inside of the small container 51 or thevicinity of the opening provided at the end of the cylindrical partserves as a first chamber in which a polymer solution 355 is put or isretained. The size of this container 305 is not particularly limited.With respect to the size of the large container 351, it has, forinstance, an inner diameter of 3 to 30 mm and a depth of 5 to 100 mm,preferably an inner diameter of 5 to 25 mm and a depth of 10 to 50 mm,and more preferably an inner diameter of 10 to 20 mm and a depth of 10to 30 mm. With respect to the size of the small container 352, the bodypart of a reverse truncated cone shape (a large volume part) has, forinstance, a maximum inner diameter of 3 to 30 mm, a minimum innerdiameter of 0.3 to 5 mm, and a height of 4 to 90 mm while thecylindrical part (the small volume part) has, for instance, an innerdiameter of 0.3 to 5 mm and a height of 0.1 to 5 mm. Preferably, thebody part of a reverse truncated cone shape (a large volume part) has amaximum inner diameter of 5 to 25 mm, a minimum inner diameter of 0.5 to3 mm, and a height of 9 to 45 mm while the cylindrical part (the smallvolume part) has an inner diameter of 0.5 to 3 mm and a height of 0.1 to3 mm. More preferably, the body part of a reverse truncated cone shape(a large volume part) has a maximum inner diameter of 10 to 20 mm, aminimum inner diameter of 1 to 2 mm, and a height of 9 to 25 mm whilethe cylindrical part (the small volume part) has an inner diameter of 1to 2 mm and a height of 0.1 to 2 mm. Furthermore, the material of thecontainer 305 is not particularly limited but for example, resin orglass can be used. With this container, when laser beam irradiation isto be carried out, the portion through which laser beam passes is formedof a transparent or semitransparent member. Examples of such a membercan include those described before. This container 305 is used asfollows, for example.

That is, first, a reservoir solution 354 is put into the large container351 (the chamber 353) and the small container 352 is filled with animmiscible hyperbaric solution 356. A magnet stirring element 307 isplaced in the bottom of the small container 352. A droplet of a polymersolution 355 is placed on the end of the cylindrical part of the smallcontainer 352. In this state, the container 305 is placed on a magnetstirrer 306 and the stirring element 307 is rotated. Thereby thegeneration of water vapor from the polymer solution 355 is promoted bythe effect of the reservoir solution 354. Furthermore, the stirringelement 307 rotates to stir the immiscible hyperbaric solution 356. Thevibration generated thereby also is transmitted to the polymer solution355 and accordingly, the polymer solution 355 also is stirredindirectly. As a result, the generation of crystalline nuclei ispromoted. When crystalline nuclei of the polymer solution have beengenerated, they may be grown thereafter and thereby intended crystalsmay be obtained. On the other hand, when no crystalline nuclei have beengenerated, the conditions employed thereby are judged to be unsuitableas the crystallization conditions and crystallization may be attemptedunder the next conditions. Further, after being brought into asupersaturation state, the polymer solution 355 may be irradiated with alaser beam. Moreover, a plurality of containers according to thisexample may be formed in one plate. The conditions thereof are the sameas in, for instance, the plate according to Example 10.

INDUSTRIAL APPLICABILITY

As described above, the process of the present invention is useful forscreening or producing a crystal of an organic substance such asprotein.

1. A process for producing a crystalline nucleus, wherein the crystalline nucleus is generated by irradiating a solution in which a solute to be crystallized has dissolved, with at least one pulsed laser selected from a picosecond pulsed laser and a femtosecond pulsed laser.
 2. The production process according to claim 1, wherein the crystalline nucleus is generated by focusing the pulsed laser in the solution with a lens and causing a local explosion phenomenon once or more in a position on which the pulsed laser is focused.
 3. The production process according to claim 1, wherein when the laser irradiation is carried out once, the pulsed laser has a pulse peak power of at least 5×10⁵ (watt).
 4. The production process according to claim 1, wherein when the laser irradiation is carried out once, the pulsed laser has a pulse energy of at least 60 nJ/pulse.
 5. The production process according to claim 1, wherein when the laser irradiation is carried out at 1000 pulses or more per second, the pulsed laser has a pulse peak power of at least 1×10⁴ (watt).
 6. The production process according to claim 1, wherein when the laser irradiation is carried out at 1000 pulses or more per second, the pulsed laser has a pulse energy of at least 1.95 nJ/pulse.
 7. (canceled)
 8. The production process according to claim 1, wherein the number of times the solution is irradiated with the pulsed laser is a single shot to 10 million shots.
 9. The production process according to claim 1, wherein the solution is a supersaturated solution.
 10. A process for producing a crystal, wherein a crystalline nucleus is allowed to be generated in a solution by a process according to claim 1 and then a crystal is grown thereon.
 11. The production process according to claim 10, wherein a solute to be crystallized is an organic substance.
 12. The production process according to claim 10, wherein a solute to be crystallized is protein. 13-25. (canceled)
 26. The production process according to claim 10, wherein a container including the solution is allowed to make a movement to stir the solution and thereby the crystal is generated and grown.
 27. The production process according to claim 26, wherein the movement is a movement selected from rotation, vibration, and rocking or a movement in which two or more of them are combined together.
 28. The production process according to claim 26, wherein the container is a well plate including a plurality of wells, and each of the wells contains the solution.
 29. The production process according to claim 26, wherein the solution is brought into a supersaturation state by evaporating a solvent contained in the solution or changing temperature of the solution.
 30. The production process according to claim 26, wherein a liquid with a higher specific gravity than that of the solution is put in the container, and the crystal is grown at an interface between the liquid with a higher specific gravity and the solution.
 31. The production process according to claim 26, wherein another container is prepared that contains a reservoir solution in which components other than the solute of the solution have dissolved at higher concentrations than in the solution, and then a crystal of the solute is generated and grown in a state where water vapor can move between the another container and the container including the solute. 32-44. (canceled) 